Abstract The prevention of childhood obesity is a key global health priority as obesity is a major contributor to increased morbidity and mortality, and is exponentially increasing in prevalence world-wide. Family aggregation studies demonstrate that adiposity tends to run in families with the heritability estimates for obesity as high as >0.70. Intergenerational studies show that ancestral nutrition can perpetuate its influence into the first and second generation of offspring via alterations in father?s sperm microRNA (miRNA) content and global methylation of DNA. While the majority of studies on intergenerational inheritance have focused on changes in parental diet, several new lines of evidence indicate that physical activity is another critical epigenetic modifier. Our published and preliminary findings demonstrate that offspring from fathers exposed to a long-term preconception exercise develop a ?thrifty phenotype?. As a result, these offspring show higher metabolic efficiency and an increased risk for obesity on a high fat diet. The observed intergenerational transmission was associated with alterations in DNA methylation and miRNA content in paternal spermatozoa. Based on these data, we propose to test the hypothesis that paternal long-term exercise induces heritable modifications in the metabolic phenotype/mitochondrial efficiency of the offspring via the male germ line. We will further determine whether these heritable changes are linked to alterations in miRNA content and DNA methylation. The specific aims for this project are: 1) To determine the impact of paternal long-term exercise on female and male offspring metabolic phenotype, mitochondrial efficiency, and susceptibility to developing obesity and glucose/insulin intolerance; 2) To determine the paternal-lineage DNA methylation patterns associated with offspring metabolic efficiency by high-throughput reduced representation bisulfite sequencing (RRBS) on epididymal spermatozoa from F0-F2 generations; 3) To determine the paternal-lineage miRNA profile associated with offspring metabolic efficiency and susceptibility to high fat diets by genome-wide RNA- Sequencing (RNA-Seq) on epididymal spermatozoa from F0-F2 generations. Integrated analysis on genome- wide RRBS methylation data and RNA-Seq data will be performed to identify cooperative relationships between methylation, miRNAs and transcriptomic changes and to further delineate the epigenetic network associated with intergenerational transmission of the thrifty phenotype. This project is expected to significantly advance our understanding of the epigenetic origins of intergenerational programming underlying familial predisposition to obesity and related metabolic diseases as well as provide excellent research opportunities for undergraduate and graduate students.